Conifers have evolved sophisticated terpenoid defenses for protection against herbivores and pathogens. Pinus massoniana Lamb. is the most widely distributed pioneer afforestation and resin tree species in China, but is seriously harmed by pine wilt disease. Monochamus alternatus is the main vector of pine wilt disease in China. Monoterpenes, sesquiterpenes and diterpenes, the main secondary defensive compounds of P. massoniana, are catalyzed by different terpene synthases (TPSs), which participate in the important defense pathways against external biotic and abiotic stresses. Here, we aimed to identify the terpene synthases (TPSs) in P. massoniana, responding to the feeding of M. alternatus, and to characterize the functions and products of the mono-TPSs. We identified six differentially expressed TPS genes in the P. massoniana fed upon by M. alternatus, including four mono-TPS and two sesqui-TPS genes. The functions of the four mono-TPSs were verified by analysis of the main product and by-products of these mono-TPSs. (+)-α-Pinene, (−)-α-pinene, and limonene were the major products of TPS (+)-α-pinene, TPS (−)-α-pinene, and TPS limonene, respectively, but TPS (−)-β-pinene only catalyzed a trace amount of (−)-β-pinene in the products. Our findings shed light on the potential relationships between the structure of terpene synthases and their corresponding products.

Background: Pinus massoniana is a significant lipid-producing tree species in China and a susceptible host for both the pine wood nematode and its insect vector, Monochamus alternatus. The basic helix–loop–helix (bHLH) family of transcription factors play a crucial role in responding to both biotic and abiotic stresses. However, the role of bHLH in terpene-induced defense in P. massoniana remains poorly studied. Results: Transcriptome sequencing using DNA Nanoball Sequencing (DNBSEQ) and PacBio Sequel platforms was performed, revealing differences in gene expression in P. massoniana branch under the simulated feeding treatment of methyl jasmonate (MeJA) spraying. Fifteen bHLH genes were cloned and analyzed, among which eight highly upregulated PmbHLH genes showed similar temporal expression after MeJA treatment and M. alternatus adult feeding. Five highly upregulated bHLH genes with nuclear localization were highly expressed in P. massoniana after M. alternatus feeding and interacted with the promoter of the terpene synthase gene Pm TPS (−)-α-pinene, confirming their involvement in the defense response of P. massoniana against the M. alternatus adult feeding. Conclusions: Our results unveil the temporal changes and the regulation of the induced defense system in P. massoniana mediated by both MeJA signaling and M. alternatus feeding treatment. The potential application for transgenic experiments and the breeding of resistant species in the future were discussed.

This study characterizes the chemical defense system of the invasive longhorn beetle Aromia bungii, a destructive pest of Prunus trees, addressing the limited understanding of chemical defensive mechanisms in Cerambycidae. High-speed cameras, environmental scanning electron microscopy (ESEM), dissection, and micro-CT imaging were used to investigate defensive behavior, and the structure of the defense system, in this beetle. Both sexes of A. bungii possess a pair of triangular, sac-like defensive glands symmetrically located in the metathorax, attached to the metasternum. Upon mechanical stimulation, white liquid defensive substances are rapidly ejected through a pair of slit-shaped openings (~200 µm) at the metasternum corners, without gland eversion, reaching over 50 cm. The average weight of substances ejected in first sprays was 7.95 ± 0.79 mg for females and 8.62 ± 2.13 mg for males (mean ± se), with no significant difference between sexes. However, the weight in second sprays after 10 days was significantly lower, at 2.93 ± 0.54 mg for females and 2.22 ± 0.40 mg for males (mean ± se), suggesting that the beetles cannot re-synthesize the substances soon after spray. The weight of ejected substances had no correlation with beetle body weight. Our findings represent the first detailed morphological and functional description of a chemical defense system in Cerambycidae, revealing a specialized metasternal gland and spray mechanism. The substantial but likely non-renewable defensive substances reflect an adaptive trade-off in energy allocation between reproduction and defense in this species that exhibits high fecundity but a short lifespan at the adult stage.